In 2014, this project provided state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed and validated and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, DNA microarray, protein separation, mass spectrometry, peptide synthesis, protein sequencing, and macromolecular X-ray crystallography. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio. Flow Cytometry The Flow Cytometry Section (FCS) provides the DIR with application-specific flow cytometric technologies, including instrumentation for high speed cell sorting and multi-color analysis. In addition, the FCS provides training, consultation, method development, and analysis for experiments involving flow cytometry. This mission is accomplished through the efforts of staff with extensive flow cytometry experience and with the use of eight cell sorters and eleven analyzers. Major goals are to provide access to state-of-the art technologies, to help design and run experiments, to facilitate data interpretation and to provide results that are of consistent high quality. Light Microscopy The Biological Imaging Section (BIS) makes advanced light microscopy imaging technologies and expertise in their use available to DIR scientists. This involves collaborating in experimental design, instrument operation and increasingly-sophisticated image processing routines to best utilize the power of these cutting edge technologies. These activities especially benefit those investigators whose specialty is not in microscopy or imaging. In practice, BIS works collaboratively with laboratories within the DIR. Investigators participate in collection of their data, either as direct microscope operators or by selecting cells of interest, so that the staff can then collect the data. Generally the collaboration begins much earlier, at the time of experimental design. The BIS advises on the most suitable instrument, appropriate labels, sample preparation, and best sampling regimens. The Section advises on the most suitable instrument, appropriate labels, sample preparation, and best sampling regimens. Another responsibility of the BIS is to anticipate new directions in the imaging requirements of DIR and to insure that it is well-equipped and knowledgeable in those areas. The section also provides software and expertise for image analysis. The facility staff advises on the correct use of the most appropriate analysis modules, as well as the best format for displaying the data. Frequently staff works with investigators to prepare images for publication including still images, video sequences, 3D reconstructions and time-lapse animations. As technology advances, image deconvolution has become more and more important as well. Protein Chemistry The Protein Chemistry Section develops project specific applications in the fields of peptide synthesis, N-terminal (Edman) sequencing, protein separation, assay development, and mass spectrometry. The RTB designs qualitative and quantitative physical-chemical and biochemical methods of detection and analysis that are customized to meet the specific research needs of DIR investigators. The Branch is also active in formulating and executing purification strategies for various types of molecules. This can also include customized small scale sample preparation and enrichment strategies for analysis by mass spectrometry. Another major activity of the RTB involves the development of more efficient and effective methods of sample preparation with a strong orientation towards mass spectroscopy to facilitate protein identification work. The Branch collaborates with DIR investigators to develop and validate protocols for protein separation and analysis for specific research programs of interest to the intramural research program. The Mass Spectrometry Facility provides support in the planning and execution of mass spectrometry (MS) projects and cooperates with other labs within the RTB to support analysis and characterization of peptides, proteins, and small bio-molecules. The facility offers all facets of mass spectrometry projects including experimental design, separation of biomolecules, sample preparation, data collection and analysis/interpretation of data. The MS facility focuses on analyzing proteins and peptides and characterizes small molecules upon request. Genomic Technologies The Genomic Technologies Section (GTS) provides state-of-the-art microarray and next-gen sequencing applications and other massively parallel genomic technologies to intramural investigators. In addition to managing high-end instrumentation, computers, and protocols involved in modern genome-wide assays, the GTS offers statistical analysis and bioinformatics to help with data interpretation. This support involves collaborating with the investigators in both experimental design and data analysis to best utilize the power of these applications, as well as to convey a better understanding of the technology. Structural Biology The Structural Biology Section (SBS) provides specialized techniques and scientific expertise that enable DIR scientists to obtain structural and biophysical information for macromolecules. Through close collaboration with DIR researchers, the SBS determines X ray crystal structures of proteins and protein-ligand complexes. X-ray crystallography requires expertise in producing large amounts of high-purity correctly folded proteins, in preparing diffraction-quality crystals, and in determining crystal structures by X-ray methods. The SBS provides consulting, produces proteins, performs biochemical and biophysical techniques, and determines structures of proteins and other macromolecules that are central to the infectious disease and immunology research programs of the DIR. The Section emphasizes providing training in biochemical, biophysical, and structural methods to investigators who can then make use of structurally-based ideas and techniques in their own laboratories.